Legionella bacteria are ubiquitous in wet or moist environments such as soil and non-marine aquatic habitats. They can also be found in warm and cold water installations, cooling towers of air conditioning systems and water humidifiers.
Legionella, especially Legionella pneumophila, are pathogens that can cause an acute bacterial pneumonia, generally known as “legionnaires disease”, which is often lethal for infected individuals.
Traditionally detection and enumeration of Legionella pneumophila are achieved by cell culturing. This method may be achieved by measuring culturable bacteria using plate count or measuring micro-colonies employing a filter membrane method. These techniques evaluate viable bacteria by their ability to form a colony or micro-colony. Unfortunately, such methods usually require between 3 and 10 days in order to allow the colonies or micro-colonies to form. Where water installations are still in operation there is an unacceptable risk of human infection during this time.
Other methods for detecting total Legionella microorganisms include PCR (Polymerase Chain Reaction) techniques. PCR employs DNA polymerase to amplify a piece of DNA by in vitro enzymatic replication. During the progression of the technique the DNA generated is used as a template for replication which brings about a chain reaction in which the DNA template is exponentially amplified. PCR enables a single or few copies of a piece of DNA to be amplified by generating millions or more copies of the DNA piece. Typically such a method is described by Diederen et al., J Med. Microbiol. 2007 January; 56 (Pt 1):94-101.
However a drawback of PCR is that the samples tend to contain polymerisation reaction inhibitors and therefore do not consistently provide quantitative results. Furthermore, the technique relies upon a prior DNA purification step which can result in loss of DNA with the consequential underestimation of the Legionella present. To some extent these disadvantages are overcome by real-time PCR which is quantitative. However, the technique cannot distinguish between viable cells and non-viable cells.
Another technique is fluorescent in situ hybridisation (FISH) in which an oligonucleotidic probe labelled by a fluorescent substance penetrates into the bacteria cells. Where the ribosomal nucleic acids (rRNA) have the correct sequence to the probe known as the target, the probe will attach itself to its target and will not be removed by any subsequent washing step. The bacteria in which the probe is fixed will then emit a fluorescent signal. This fluorescent signal may then be quantified by techniques such as flow cytometry, solid phase cytometry, or epifluorescent microscopy. A typical FISH technique is described by Dutil S et al J Appl Microbiol. 2006 May; 100(5):955-63. However, using the FISH technique alone the total number of viable Legionella pneumophila could be detected but unfortunately the method could not exclusively identify only those Legionella pneumophila bacteria able to divide and by consequence make a colony.
A further method for enumerating viable Legionella pneumophila involves ChemChrome V6 and is described by Delgado-Viscogliosi et al Appl Environ Microbiol. 2005 July; 71(7):4086-96. This method allows the quantification of Legionella pneumophila as well as discrimination between viable and non-viable bacteria. It combines specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6) and employing epifluorescent microscopy for the enumeration. However, although this technique distinguishes between viable and non-viable cells it is not able to separately identify those colony-forming bacteria.
US 20070218522 describes methods and compositions for detecting and quantifying viable Legionella and other heterotrophic aerobic bacteria the method includes the use of dipslides that include an absorbent medium, growth promoting and growth selective substances for rapid detection and quantification of micro-colonies of Legionella. This technique would not enumerates injured bacteria.
EP 1329515 relates to a method of testing for the presence of microorganisms in a gaseous environment comprising hydrogen peroxide by bringing the gaseous environment into contact with an agar growth medium comprising a salt of pyruvic acid and allowing the development of colonies of the microorganisms.
Techniques which involve the growth of colonies on a growth medium, such as a nutrient agar plate, are generally considered to be more accurate. Consequently the plate count method remains the preferred choice of method for obtaining the total viable count. This generally means applying a sample suspected of containing the microorganism onto a plate containing a solid nutrient source or growth medium. Such a technique is generally referred to as plating. By total viable count we mean the total number of bacteria capable of yielding a population discernible by the observer. Typically this will mean a visible colony on the surface of a growth medium such as nutrient agar plate.
However, microorganisms such as Legionella pneumophila in the environment may be subject to one or more stresses which prevent the microorganism from growing and multiplying in its environmental situation. Such stressed microorganisms would not divide at all or form a visible colony under normal culturing conditions. In the environment a proportion of microorganisms cells will generally be stressed due to environmental conditions, such as starvation, presence of biocide, heat shock and desiccation. Furthermore, these cells may be in a vulnerable physiological state in which the technique of plating the microorganisms may exacerbate stressing of those already stressed microorganisms cells due to the presence of atmospheric oxygen. Furthermore this could lead to artifactual death of the stressed bacteria leading to an underestimation of the total viable count.
In addition, underestimation of viable Legionella pneumophila with plating method might become hazardous in regard to its pathogenicity.
Since the 1970s it has been reported that scavengers of reactive oxygen species (ROS) should be used to limit the effect of oxidative stress during the plating process. This was reported by Speck et al, repair and enumeration of injured coliforms by a plating procedure, Appl Microbiol 29, 549-50 (1975); Martin et al Catalase: its effect on microbial enumeration. Appl Environ Microbiol 32, 731-4 (1976); Brewer et al Beneficial effects of catalase or pyruvate in a most-probable-number technique for the detection of Staphylococcus aureus. Appl Environ Microbiol 34, 797-800 (1977); McDonald et al, Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation. Appl Environ Microbiol 45, 360-5 (1983); Marthi et al) Resuscitation effects of catalase on airborne bacteria. Appl Environ Microbiol 57, 2775-6 (1991); Busch and Donnelly Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua. Appl Environ Microbiol 58, 14-20 (1992); and Dukan et al, Oxidative stress defense and deterioration of growth-arrested Escherichia coli cells. J Biol Chem 274, 26027-32 (1999).
However, in all the aforementioned cases the inventors of the present invention believe that the ROS would be reduced by a direct route in which the compound reacts chemically with ROS.
Bérubé et al, “Rapid detection and identification of Legionella pneumophila by membrane immunoassay”, Applied and Environmental Microbiology, 1989, 55, 1640-1641 describes the detection and identification of Legionella pneumophila by an immunoblot assay using a monoclonal antibody. No means is provided for dealing with the problem of injured bacteria.
An article by Pine et al (Role of keto acids and reduced-oxygen-scavenging enzymes in the growth of Legionella species. J Clin Microbiol 23, 33-42 (1986)) describes the necessity for the addition of keto acids and reduced oxygen scavenging enzyme is to optimise the growth of Legionella pneumophila and suggested using these materials in the medium used for standard enumeration of this microorganism.
However the use of keto acids and reduced oxygen scavenging enzyme alone is insufficient to repair the stressed Legionella pneumophila cells to be repaired and allow accurate enumeration. This is especially so when using a specific growth medium for Legionella pneumophila, such as buffered charcoal yeast extract (BCYE) agar medium. In fact, there is no data available concerning the optimisation of a standard medium useful for the accurate enumeration of Legionella pneumophila. 
Thus an objective of the present invention is to find a method for accurately enumerating Legionella pneumophila. This is especially so in regard to its standard method using the plating technique.